The long range objective of this research is to establish a detailed description of the metallo-biochemistry of the essential trace metal, copper, in the yeast, Saccharomyces cerevisiae. The significance of this research is that there is limited mechanistic information about the cellular biochemistry of any of the essential divalent transition metal ions in any eukaryote. This proposal is designed to test two specific aspects of a model of the uptake and cellular utilization of Cu in S. cerevisiae based on published and preliminary work described in this proposal. These two aspects are: 1) that uptake of Cu(II) from the medium involves Cu(II) reduction to Cu(I) catalyzed by a plasma membrane reductase activity and 2) that glutathione is a component of the intracellular trafficking of the Cu(I) which is taken into the cell. Four Specific Aims are described which address these aspects and which will also provide additional genetic reagents to extend these studies in the future. Aim I. Clone and characterize the mutant allele, cup3, which exhibits kinetically faster Cu accumulation and a correspondingly elevated Cu(II) reductase activity. The proposed model is largely based on our preliminary studies of this mutation. Aim II. Demonstrate that reduction of medium Cu(II) to cell-associated Cu(I) is a possible step in Cu accumulation by S. cerevisiae, that this activity is represented by the Cu(II) reductase activity, and biochemically characterize this activity. Aim III. Determine the possible function(s) of glutathione (GSH), in the redistribution of Cu in the cytosol of S. cerevisiae, and the role of Cu-thionein as a functional Cu store for the activation of apo-Cu,Zn superoxide dismutase (SOD-1). Aim IV. Isolate new mutants which exhibit slower or aberrant Cu accumulation which may include mutants in Cu(II) reduction, transport, or intracellular trafficking. These will serve as the basis for future tests of the model, in particular, to test the possibility that the Cu(II) reductase and putative Cu-transporter are the same gene product and to confirm the role of specific intracellular factors in Cu-handling. The experimental design is based on the unique characteristics of S. cerevisiae among eukaryotes which include well-established classical and molecular genetics and an ease of systematic and controlled in vivo manipulation and analysis. Most of the reagents needed to begin this work have been prepared. The details of copper metallobiochemistry which emerge from these studies will provide a paradigm for possible mechanisms of Cu-handling in higher eukaryotes.